TPA-induced miR-101 and its downstream effects are all PKCα-dependent in HepG2 cells. (A) HepG2 and HepG2 PKCα knockdown cells were treated with 100 nM TPA for 48 hours. Expression levels of miR-101 was normalized to miR-16 and expressed as fold change using DMSO-treated sample as baseline. (B) Alignment of miR-101 sequence and the predicted miR-101 target sites in the 3'UTR of EZH2 and EED. (C) HEK293 and HepG2 cells were co-transfected either pMIR-REPORT constructs of EED-3'UTR(+1~+ 211) and EZH2-3'UTR(+1~+263) 10 ng with 4 μg of miR-LacZ, miR-101-1, and miR-101-2 for 48 hours. Forty-eight hours after transfection, cells were harvest for luciferase activity analysis. Results shown are averages of three independent experiments performed in triplicate. (D) HepG2 and HepG2 PKCα knockdown cells were treated with 100 nM TPA for 48 hours. Cell lysates were collected for immunoblotting assay using the PKCα, EZH2, SUZ12, EED, p21, and H3K27me3 antibodies.